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研究生:黃獻慶
研究生(外文):Hsien-ChingHuang
論文名稱:在腦下垂體腫瘤細胞 (GH3)中美金剛胺 (Memantine)與細胞膜上膽固醇含量的變動對電穿孔所引發向內電流的影響
論文名稱(外文):Effects of memantine and cholesterol content on electroporation-induced currents in pituitary tumor GH3 cells
指導教授:吳勝男
指導教授(外文):Sheng-Nan Wu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:39
中文關鍵詞:美金剛胺膜上膽固醇膜穿孔電流腦下垂體腫瘤細胞
外文關鍵詞:membrane electroporationmemantinemembrane cholesterolinward currents
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細胞膜電穿孔技術 (Membrane electroporation)在研究中被廣泛的熟知,能夠藉由外在給予細胞一個電場的刺激,而使得細胞膜的電導性與通透性得到明顯的增加。由於細胞上跨膜電位的增加 (transmembrane potential),科學家們研究指出在細胞膜上會因此產生奈米級的孔洞 (nanoscale pores),進而允許分子自由的進出細胞。美金剛胺 (Memantine),為金剛烷胺類 (amantadine)的衍生物,長久以來是被公認的有效抗病毒藥劑。根據之前的研究指出,細胞膜上膽固醇總量的變化也可能會改變膜上離子通道的活性和細胞膜的特性。然而,對於細胞膜穿孔後所引發的電流其電學與藥物學特性,仍然存在著很多疑問。因此,我們利用膜片箝技術 (patch clamp)來研究細胞膜穿孔後所引發的膜電穿孔電流 (IMEP)的特性。當細胞膜受到過極化刺激,短暫和不規則形狀的內向電流 (IMEP)會明顯的被誘發。加入氯化鑭 (LaCl3-) 300 μM和美金剛胺 300 μM後,在研究中都發現能對這種電流的大小產生抑制作用。在電生理實驗中使用完整細胞紀錄,發現美金剛胺並不會對於受電壓調控而活化的鈣離子通道 (voltage-gated Ca2+ current)活性造成任何抑制的作用。應用內翻外模式紀錄,在腦下垂體前葉腫瘤細胞加入美金剛胺,發現不論是在胞內或胞外,都不會去抑制受到三磷酸腺苷 (ATP)所活化的鉀離子通道 (ATP-sensitive K+ currents)活性表現。在另外一系列的實驗中,實驗結果顯示出細胞經由 (2-羥丙基)--環糊精 ((2-hydroxypropyl)--cyclodextrin)處理後使得細胞膜上的總膽固醇量減少,導致細胞膜穿孔後所引發的電流 (IMEP)密度減少,加入氧化物的誘發劑進而去提高此種電流的幅度增益效果也受到中止。我們的實驗結果證實了在腦下垂體前葉腫瘤細胞中,美金剛胺會去抑制細胞膜穿孔後所引發的電流,進而使電流幅度減少。但美金剛胺在腦下垂體前葉腫瘤細胞中去抑制此種電流的調節機制,和其去抑制由於穀氨酸 (glutamate)誘發而產生的電流,或是因三磷酸腺苷 (ATP)活化的鉀離子電流其機制並不相關聯。使用 (2-hydroxypropyl)--cyclodextrin 去減少膜上膽固醇的總量可能會影響膜上孔洞的形成,因此去減少了細胞膜穿孔後所引發的電流大小。因此我們認為美金剛胺與細胞膜上膽固醇總量的變化對於此電流的抑制效果可能會與細胞的功能有關。
Membrane electroporation (MEP) is recognized to exert an increase in the electrical conductivity and permeability of the plasma membrane by use of an externally applied electrical field. Owing to an increase in the transmembrane potential, nanoscale pores can form in the cell membrane, thus allowing molecules to be transported into and out of the cells. Memantine (MEM), a derivative of amantadine, is recognized as an antiviral agent. Previous study indicates that changes in the amount of membrane cholesterol can directly modify the activity of membrane ion channels and membrane properties. The electrical and pharmacological properties of MEP-induced inward current (IMEP) remain largely unknown. In this study, we attempted to use the patch-clamp technique to investigate the properties of IMEP. When cell membrane hyperpolarized, an irregular and transient inward current (IMEP) was clearly elicited. Either LaCl3- (300 μM) or MEM (300 μM) could block this current. In whole cell recording, MEM did not exert any effect on voltage-gated Ca2+ current. In inside-out configuration, MEM applied to either external or internal surface of the excised patch did not suppress the activity of ATP-sensitive K+ channels expressed in GH3 cells. In another series of experiments, our data was showed that depletion of membrane cholesterol by exposing cells to HPCD resulted in reduction of IMEP density. Our study provides the first evidence to show that MEM directly inhibits the amplitude of IMEP in pituitary GH3 cells. MEM-mediated block of IMEP in these cells is unlinked to its inhibition of glutamate-induced currents or ATP-sensitive K+ currents. Deplete of membrane cholesterol by HPCD can alter the pore formation, thereby decreasing the IMEP magnitude. The inhibition of MEM and depletion cholesterol in IMEP might be related to cell function.
中文摘要 III
Abstract V
Acknowledgement VII
Introduction 1
Materials and methods 6
Cell preparations 6
Drugs and solutions 6
Electrophysiological measurements 7
Data recordings and analyses 8
Results 10
Electrical properties of IMEP in pituitary GH3 cells 10
Single channel properties of IMEP in pituitary GH3 cells 10
Concentration-dependent inhibition of LaCl3 and memantine on the amplitude of IMEP 11
Inhibitory effects of LaCl3 and MEM on the current-voltage (I–V) relationships of IMEP 11
Lack of MEM effect on ICa in GH3 cells 12
Inability of MEM to block KATP channels in GH3 cells 12
Effect of HPCD treatment on the density of IMEP in pituitary GH3 cells 13
Effect of 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) in GH3 cells treated or untreated with HPCD 14
Effect of HPCD treatment on I-V relationship of IMEP in GH3 cells 14
Discussion 16
Figure Legends 19
Figures 24
References……………………………………………………………………...34

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